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Novel Anti-cd3epsilon antibodies

Active Publication Date: 2020-09-24
WUXI BIOLOGICS IRELAND LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present patent provides a way to modify an antibody or antigen-binding fragment thereof, which can improve its binding affinity and enhance or reduce its ability to activate different immune responses. This can be useful for developing pharmaceutical compositions that can be used to treat disease or reduce side effects.

Problems solved by technology

Although OKT3 has strong immunosuppressive potency, its clinical use was hampered by serious side effects linked to its immunogenic and mitogenic potentials (Chatenoud (2003) Nature Reviews 3:123-132).
Such serious side effects limited the more widespread use of OKT3 in transplantation as well as the extension of its use to other clinical fields such as autoimmunity (Id.).

Method used

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  • Novel Anti-cd3epsilon antibodies

Examples

Experimental program
Comparison scheme
Effect test

example 1

n of Hybridoma Antibody

1.1 Animal Immunization

[0275]Recombinant extracellular domains (ECD) proteins of human CD3, including human CD3 epsilon (CD3epsilon) with His Tag (cat. No.: 10977-H08H; Sino Biological Inc. Beijing, China), human CD3 Gamma (CD3gamma) (cat. No.: ab140563; Abcam Shanghai China) and human CD3 delta (CD3delta) with His Tag (cat. No.: 10977-H08H; Sino Biological Inc. Beijing, China) were used as immunogens for animal immunization. Balb / c mice were purchased from Shanghai SLAC laboratory animal Co, Ltd. and were housed in an IACUC approved animal facility. Mice were immunized with the ECD protein mixture of CD3epsilon, CD3gamma and CD3delta or immunized with 7×106 freshly isolated human T-cells for each mouse, mixed with the Titermax adjuvant by subcutaneous and footpad injections every other week.

[0276]Blood were collected from mice before and after immunization and serum titers against target proteins were monitored by ELISA.

1.2 Hybridoma Generation

[0277]The mouse...

example 2

n of Humanized Antibody

2.1 IgG Conversion and Humanization

[0282]Generation of Chimeric Antibody from Marine-Derived mAbs:

[0283]WBP3311_2.166.48 and WBP3311_2.306.4 and WBP3312_3.179.16 VH and VL genes were re-amplified with cloning primers containing appropriate restriction sites and cloned into WuXi Biologics' proprietary expression vector to create corresponding clones of chimeric antibodies with constant region of human IgG1.

[0284]Humanization and Synthesis of Humanized V-Genes:

[0285]“Best Fit” approach was used to humanize WBP3311_2.166.48 and WBP3311_2.306.4 light and heavy chains. For light chains amino acid sequences of corresponding V-genes were blasted against in-house human germline V-gene database. The sequence of humanized VL-gene was derived by replacing human CDR sequences in the top hit with mouse CDR sequences using Kabat CDR definition. Frameworks were defined using extended CDR where Kabat CDR1 was extended by 5 amino acids at N-terminus. Multiple humanized sequenc...

example 3

Characterization

3.1 Antibody Binding by ELISA and FACS

[0288]Antigens, antibodies and cells used in the ELISA and FACS described below are listed in table 3.

TABLE 3Antigens, antibodies and cells used in ELISA and FACSMaterialCompanyCat. log No.Human CD3 epsilon (CD3epsilon)Sino Biological Inc.10977-H08Hprotein His Tag)Human CD3 delta (CD3 delta) proteinSino Biological Inc.10981-H08H(His Tag)Human CD3 Garmma (CD3gamma)Abcamab140563proteinCynomolgus Monkey CD3 epsilonACROCDE-C5226proteinCyno PBMCsPharmaLegacy Laboratories(Shanghai) Co.Mouse anti human CD3 epsilonSino Biological Inc.10977-MM03monoclonal antibody(Clone NO.:1A7E5G5)Mouse anti-human CD3 delta monoclonalSino Biological Inc.10981-MM08AntibodyMouse anti-human CD3gammaSANTA CRUZsc-55563monoclonal AntibodyBIOTECHNOLOGY, INCBenchmark antibody OKT3Abcamab86883Jurkat cellsATCCTIB-152HUT78 cellsATCCECACC-880401901MOLT-4 cellsATCCCRL-1582Semi-solid mediumStemcells03814

[0289]Binding of Antibodies to Protein by ELISA and EC50:

[0290]Hu...

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Abstract

The present disclosure provides isolated monoclonal anti-CD3epsilon antibodies or antigen-binding fragments thereof comprising one or more heavy chain CDR sequences selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 47, and / or one or more kappa light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 48, isolated polynucleotides encoding the same, pharmaceutical compositions comprising the same, and the use thereof.

Description

FIELD OF THE INVENTION[0001]The present disclosure generally relates to novel anti-human CD3epsilon antibodies.BACKGROUND[0002]The CD3 (cluster of differentiation 3) T-cell co-receptor is a protein complex and is composed of four distinct chains, a CD3gamma chain, a CD3delta chain, and two CD3epsilon chains. These chains associate with a molecule known as the T-cell receptor (TCR) and the zeta-chain to generate activation signal in T lymphocytes. The TCR, zeta-chain, and CD3 molecules together comprise the TCR complex, in which TCR as a subunit recognizes and binds to antigen, and CD3 as a subunit transfers and conveys the antigen-stimulation to signaling pathway, and ultimately regulates T-cell activity. The CD3 protein is virtually present in all T cells.[0003]The CD3 together with TCR forms a CD3-TCR complex, which plays pivotal role in modulating T cell vast functions in both innate and adoptive immune response, as well as cellular and humoral immune functions. These include eli...

Claims

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Application Information

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IPC IPC(8): C07K16/28A61K47/68
CPCC07K16/2809C07K2317/24C07K2317/565A61K47/6803C07K2317/31C07K2317/77C07K2317/92C07K2317/567C07K2317/33A61P35/00G01N33/74G01N2333/7051A61K2039/505
Inventor LI, JINGMEI, QIN
Owner WUXI BIOLOGICS IRELAND LTD
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